CN102283113B

CN102283113B - Method for constructing sweet sorghum high-frequency regeneration system by using young ear as explant

Info

Publication number: CN102283113B
Application number: CN 201110168768
Authority: CN
Inventors: 边鸣镝; 邓川; 杨振明
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2011-06-22
Filing date: 2011-06-22
Publication date: 2013-03-27
Anticipated expiration: 2031-06-22
Also published as: CN102283113A

Abstract

The invention relates to a method for constructing a sweet sorghum high-frequency regeneration system by using a young ear as an explant, and belongs to the technical field of plant gene engineering. The method comprises: cutting a 2-to-3-centimeter young ear of sweet sorghum in a flag leaf period into sections; inoculating the sections onto an induction culture medium for culture and obtaining calluses; performing subculture, differentiated culture and root induction; and transplanting in a green house or field. In the invention, the young ear is proved to be an ideal explant for constructing a sweet sorghum regeneration system and can be used for further researches on transformation.

Description

A kind of method of the constructing sweet sorghum high-frequency regeneration system take young fringe as explant

Technical field

The present invention relates to the plant gene engineering technology field, particularly relate to a kind of method of the constructing sweet sorghum high-frequency regeneration system take young fringe as explant.

Background technology

Sugar grass (Sorghum bicolor (L.) Moench) claim again sugared Chinese sorghum, it is the mutation of common grain sorghum, have the characteristics such as drought resisting, waterlogging, impoverishment tolerant, Salt And Alkali Tolerance, suit to plant at marginality soil such as saline and alkaline arids, simultaneously, the sugar grass growth is fast, output is high, stem stalk sugar juice rich content, therefore, be described as " the strongest competitor in the bioenergy system " (Antonopoulou et al.2008; Carpita et al.2008).Sugar grass is as the renewable sources of energy crop of tool advantage, and (Rooney 2004 to have caused extensive concern; Farrell, 2006).2009, the mechanisms such as Polymorphism group research institute of USDOE finished grain sorghum gene order-checking and analytical work (Andrew et al.2009), and this has greatly promoted the further investigation of sugar grass molecular genetics.At present, in the urgent need to excavating by transgenic technology and identify excellent degeneration-resistant border stress gene, and then be applied to the directed genetic improvement of sugar grass kind.Up to now, it is less that the genetic transformation work sutdy of sugar grass is carried out scale, and technical development is slow.Therefore, the sugar grass regenerating system of setting up efficient stable is used for genetic transformation, has become the current matter of utmost importance that needs to be resolved hurrily.

Present research of cultivating for cultivating sweet sorghum tissue also seldom.Grain sorghum research relatively early provides foundation and reference for we study sugar grass.Masteller in 1970 etc. induce callus, and have obtained regeneration plant take the former base of bud as explant, and this is to cultivate the earliest report that obtains the Chinese sorghum regeneration plant by tissue.After this, the various countries scholar has carried out the research work that the Chinese sorghum tissue is cultivated in succession, now from Immature Sorghum Embryos in vitro (Dunstan et al.1979; Elkonin et al.1995; Ma et al.1987; Sairam et al.2000; Zhao et al.2000; Hagio 2002), mature embryo (Mackinnon et al.1986; Cai et al.1987), stem apex (Nahd et al.1995; Kishore et al.2006), seed (Yang et al.1990) and spire (Wernicke et al.1980; Sairam et al.1999) etc. all obtained regeneration plant on the different explants, young fringe has advantages of that differentiation rate is high, easily obtain regeneration plant, is considered to desirable explant (the Brettell et al.1980 of Chinese sorghum group training; Wen et al.1991; Kaeppler et al.1997; Mani et al.2003; Jogeswar et al.2007).

The tissue of Chinese sorghum is cultivated and is subjected to genotype to affect large (Kaeppler et al.1997; Hagio 1994; Kuruvinashetti et al.1998), lacking good acceptor gene type is slowly main cause of Chinese sorghum Study on Genetic Transformation job development.Obtaining the strong genotype of callus of induce and differentiation and regeneration ability, is to transform successful prerequisite.There is not yet so far report.

Summary of the invention:

The invention provides a kind of method of the constructing sweet sorghum high-frequency regeneration system take young fringe as explant, to be used for the Study on Genetic Transformation of sugar grass.

The technical scheme that the present invention takes is to comprise following sequential steps:

1) chooses the young fringe of sugar grass sword-like leave phase 2～3cm, be cut into segment;

2) be inoculated on the inducing culture and cultivate, form callus, inducing culture is: MS minimal medium+2,4-D 3mg/L+KT (kinetin) 0.5mg/L+30g/L sucrose+8.0g/L agar pH5.8;

3) callus that induces is transferred in the subculture medium cultivated, subculture medium is: MS minimal medium+2,4-D 1.5mg/L+KT (kinetin) 0.25mg/L+30g/L sucrose+8.0g/L agar pH5.8;

4) subculture was cultivated after 30 days, callus is transferred to be cultured in the differential medium emerges, differential medium is: MS minimal medium+IAA (heteroauxin) 1mg/L+KT (kinetin) 0.5mg/L+ ascorbic acid 10mg/L+30g/L sucrose+8.0g/L agar pH5.8;

5) regrowth is transferred to carries out inducing of root in the root media, root media is: 1/2MS medium+30g/L sucrose+8.0g/L agar pH5.8;

6) be transplanted to greenhouse or land for growing field crops.

Described young spike length degree is 2～3cm, places 2 days in-4 ℃ of refrigerators, is cut into 2-3mm long.

Condition of culture is in the described inducing culture: in 25 ± 2 ℃ of dark cultivations 30 days.

Described subculture cultivate be 7 days subcultures once.

Condition of culture is in the described differential medium: in 25 ± 2 ℃ of lower 16h/d illumination cultivation.

Described regrowth grows to when approximately 5cm is high, moves in the root media.

Described sugar grass kind is FETERITA NIDIANA, MN-2949, MN-2904, MN-3013 and SILVER TOP-1.

The present invention utilizes the sweet sorghum young fringe to be explant, adopts specific medium component to callus of induce, propagation, and differentiation, until obtain regeneration plant, the inventive method can obtain higher callus induction rate and differentiation rate.Realize that the result shows that young fringe is to set up the more satisfactory a kind of explant of sugar grass regenerating system, can be used for further Study on Transformation.

Description of drawings

Fig. 1: sweet sorghum young fringe Regeneration System schematic diagram.

A: inoculation; B: young fringe expands; C-F: the Callus morphology of different genotype; G: callus differentiation initial stage; H: seedling rooting process; I: the seedling of transplant survival.

Embodiment

The present invention is described in further detail below in conjunction with embodiment.

1) the young fringe material that is used for the sugar grass Regeneration System is selected and preparation

The present invention chooses the young fringe of sugar grass sword-like leave phase 2～3cm, places 2 days in-4 ℃ of refrigerators, with 75% ethanol explant, then outer bract is peelled off after taking out, and strips young fringe with scalpel in superclean bench, young fringe is cut into 2-3mm long;

2) be inoculated on the inducing culture, inducing culture is: MS minimal medium+2,4-D 3mg/L+KT (kinetin) 0.5mg/L+30g/L sucrose+8.0g/L agar pH5.8,25 ± 2 ℃ of dark cultivations 30 days;

3) slightly expand after being inoculated in young fringe 5d on the inducing culture, 7 days left and right sides cob base portions or Xiao Hua top begin to form callus, most of explant all induces callus behind inoculation 30d～40d, remove the callus of serious brownization, transfer in the subculture medium, subculture medium is: MS minimal medium+2,4-D 1.5mg/L+KT (kinetin) 0.25mg/L+30g/L sucrose+8.0g/L agar pH5.8,7 days subcultures once, condition of culture is 25 ± 2 ℃ of dark cultivations;

4) subculture was cultivated after 30 days, callus is transferred to differential medium, differential medium is: among MS minimal medium+IAA (heteroauxin) 1mg/L+KT (kinetin) 0.5mg/L+ ascorbic acid 10mg/L+30g/L sucrose+8.0g/L agar pH5.8,25 ± 2 ℃ of lower 16h/d illumination cultivation, intensity of illumination is about 40-45 μ mol m ^-2S ^-1

5) treat that regrowth grows to when approximately 5cm is high, be separated into single seedling, be transferred to root media, root media is: carry out inducing of root among 1/2MS medium+30g/L sucrose+8.0g/L agar pH5.8, the seedling that has taken root is shifted out from medium, clean the agar that is bonded on the root system;

6) transfer is to greenhouse or land for growing field crops.

1, genotype is on the impact of callus induction

61 kinds of Genotypes for examination have 49 kinds of genotype sugar grasses to be induced out callus, and the callus status that the sugar grass of different genotype induces is different, and some callus are white in color or are faint yellow, graininess, and growth rate is very fast; It is yellow that some callus appearances are, and quality is fine and close, fast growth, generally brownization not; Also have a kind of callus appearance to be light yellow, closely, secreting mucus, growing way a little less than, grow slower.Genotype is different, and callus induction rate is different, and FETERITA NIDIANA, MN-2949, the MN-29043 genotype ability that goes out to heal is the strongest, and callus induction rate is respectively 96.7%, 96.7%, 100%.Variance analysis (table 1) is the result show, calli induction frequencies difference reaches utmost point significance level between genotype, this shows, genotype plays an important role to inducing of callus.

The variance analysis of table 1 callus induction rate

2, genotype is on the impact of differentiation and regeneration

49 genotype sugar grasses of callus induction success are broken up and regenerate, the result shows, phenylacetic acid difference is obvious between genotype, wherein MN-3013 and SILVER TOP-1 have higher differentiation capability, differentiation rate is the highest, reaches 87.5% and 64.1%, and variance analysis (table 2) is the result show, phenylacetic acid difference reaches utmost point significance level between genotype, illustrates that genotype is a key factor that affects differentiation and regeneration.

The variance analysis of table 2 phenylacetic acid

3, browning

Browning is running through whole explant and is becoming more process.Become the more initial stage, the explant edge just begins to occur browning, and callus forms the pigment of a large amount of dark browns or purple pigment gradually, and spreads element in medium, and serious meeting causes callus downright bad.Should avoid occurring browning as far as possible in the group training, for reducing brownization, can solve by shortening Subculture Time, Subculture Time of the present invention is 7 days, has controlled preferably browning, with brownization callus Partial Resection, prevents that brownization from spreading in the subculture process; When selecting kind, also should avoid as possible the genotype of using brownization degree serious.

Conclusion: the present invention shows that callus induction rate and differentiation rate have notable difference between different genotype, the performance of the several respects such as comprehensive healing rate, differentiation capability, brownization degree, think that reacting phase that the young fringe of genotype FETERITA NIDIANA, MN-2949, MN-2904, MN-3013 and SILVER TOP-1 cultivates tissue to better, can be used as the desirable acceptor material of genetic transformation.

Claims

1. the method for the constructing sweet sorghum high-frequency regeneration system take young fringe as explant is characterized in that comprising the steps:

1) chooses the young fringe of sugar grass sword-like leave phases 2 ~ 3 cm, be cut into segment;

2) be inoculated on the inducing culture and cultivate, form callus, inducing culture is: MS minimal medium+2, and 4-D 3 mg/L+KT0.5 mg/L+ 30g/L sucrose+8.0g/L agar, pH5.8, condition of culture is: in 25 ± 2 ℃ of dark cultivations 30 days;

3) callus that induces is transferred in the subculture medium cultivated, subculture medium is: MS minimal medium+2,4-D 1.5 mg/L+KT0.25 mg/L+ 30g/L sucrose+8.0g/L agar, pH5.8;

4) subculture was cultivated after 30 days, callus is transferred to be cultured in the differential medium emerges, differential medium is: MS minimal medium+IAA 1 mg/L+ KT0.5 mg/L+ ascorbic acid 10 mg/L+ 30g/L sucrose+8.0g/L agar, pH5.8, condition of culture is: in 25 ± 2 ℃ of lower 16 h/d illumination cultivation;

5) regrowth is transferred to carries out inducing of root in the root media, root media is: 1/2MS medium+30g/L sucrose+8.0g/L agar, pH5.8;

6) be transplanted to greenhouse or land for growing field crops.

2. the method for a kind of constructing sweet sorghum high-frequency regeneration system take young fringe as explant as claimed in claim 1 is characterized in that: described 2 ~ 3 cm children fringe, in-4 ℃ of refrigerators, placed 2 days, and be cut into 2-3mm long.

3. the method for a kind of constructing sweet sorghum high-frequency regeneration system take young fringe as explant as claimed in claim 1 is characterized in that: described subculture cultivate be 7 days subcultures once.

4. the method for a kind of constructing sweet sorghum high-frequency regeneration system take young fringe as explant as claimed in claim 1 is characterized in that: described regrowth grows to 5cm when high, moves in the root media.

5. the method for a kind of constructing sweet sorghum high-frequency regeneration system take young fringe as explant as claimed in claim 1, it is characterized in that: described sugar grass kind is FETERITA NIDIANA, MN-2949, MN-2904, MN-3013 and SILVER TOP-1.